Peptide mimotope to mycotoxin deoxynivalenol and uses thereof

ABSTRACT

The present invention provides a peptide mimotope of the non-peptide mycotoxin deoxynivalenol. In particular, the peptide mimotope competes with deoxynivalenol for binding to a monoclonal antibody and is antagonistic to the inhibitory effects of deoxynivalenol on in vitro protein synthesis. The present invention also provides a method that uses the peptide mimotope to determine whether corn, grains or mixed feed is contaminated with fungi that produces deoxynivalenol. The present invention further provides transgenic plants resistant to deoxynivalenol.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Serial No.60/146,643 filed Jul. 30, 1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported by U.S. Department of Agriculture ResearchNational Research Initiative grant 9702545 and Public Health Servicegrant E5-03358. The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a peptide mimotope of the non-peptidemycotoxin deoxynivalenol. In particular, the peptide mimotope competeswith deoxynivalenol for binding to a monoclonal antibody, isantagonistic to the inhibitory effects of deoxynivalenol on in vitroprotein synthesis, and does not elicit antibodies in mice that recognizethe deoxynivalenol. The present invention also relates to a method thatuses the peptide mimotope to determine whether corn, grains or mixedfeed is contaminated with fungi that produces deoxynivalenol. Thepresent invention further relates to transgenic plants resistant todeoxynivalenol.

(2) Description of Related Art

Deoxynivalenol (DON) or vomitoxin or dehydronivalenol is12,13-epoxy-3,7,15-trichothec-9-en-8-one, which is a mycotoxin of the12,13-epoxy-trichothecenes class of sesquiterpene mycotoxins. It isproduced primarily by the fungus Gibberella zeae (Schwein.) Petch(anamorph=Fusarium graminearum Schwabe), which infects corn, smallgrains and mixed feeds (Hart et al., J. Agric. Food Chem. 31: 657-659(183); Hart et al., Plant Dis. 66: 1133-1135 (1982); Neish et al., Can.J. Plant Sci. 61: 811-815 (1981)). At the cellular level, the primarytoxic effect of DON is inhibition of protein synthesis by binding to the60S ribosomal subunit, which interferes with peptidyltransferase(Betina, Chem. Biol. Interact. 71: 105-146 (1989); Weber et al.,Biochem. 31: 9350-9354 (1992)). DON can cause anorexia and emesis inanimals (Scott et al. Proc. natl. Acad. Sci. USA 89: 5398-5402 (1992)).Other toxic effects of DON include skin irritation, hemorrhaging,hematological changes, human lymphocyte blastogenesis impairment,radiomimetic effects, apoptosis and immunotoxicity (Scott et al. ibid.).

DON is primarily found as a contaminant in grains that are infected withthe above fungi. It has also been implicated as a chemical warfareagent. Currently, the only means for eliminating DON from human andanimal foodstuffs is to detect DON in food and to remove anycontaminated foodstuffs from the food supply. Immunoassays offer severaladvantages compared to other analytical methods for detecting DON infoodstuffs. Following the development of the first monoclonal antibodyto DON (Casale et al., J. Agric. Food Chem. 36: 663-668 (1988)),immunological methods, primarily enzyme-linked immunosorbant assay(ELISA), have been widely used for detection of DON (Pestka et al., FoodTechnol. 49: 120-128 (1995)). An immunoassay for trichothecenes such asDON is disclosed in U.S. Pat. No. 4,879,248 to Chu et al. and kitcomprising the immunoassay is disclosed in U.S. Pat. No. 5,118,612 toChu et al. The immunoassay and kit are either radio immunoassays (RIA)or enzyme-linked immunosorbant assay ELISA based on a competitivecontrol that is DON. These immunological assays have advantages whichinclude high specificity, ease of use, facile sample preparation, andgood sensitivity.

The disadvantages of these immunoassays is that they require the user tohandle purified DON which poses a toxicity risk to the user. Inaddition, chemical conjugation of DON to a carrier protein or an enzymehas low efficiency because it involves extensive modification andblocking stages and causes substantial bridge-group interferences andun-wanted cross-reactions (Casale et al., ibid.: Pestka et al., ibid.;Yuan et al., Appl. Environ. Microbiol. 63: 263-269 (1997)). Furthermore,DON is poorly immunogenic and when DON is conjugated to a carrierprotein, it's immunogenicity is only marginally enhanced.

Therefore, it is desirable that an alternative to DON be developed.Preferably, the DON alternative would be non-toxic to the user, notrequire conjugation to a protein, and be highly immunogenic.

SUMMARY OF THE INVENTION

The present invention provides a peptide mimotope of thenon-proteinaceous mycotoxin deoxynivalenol (DON). In particular, thepeptide mimotope competes with DON for binding to a monoclonal antibodyagainst the DON, is antagonistic to the inhibitory effects of DON on invitro protein synthesis, and does not elicit antibodies in mice thatrecognize DON.

The peptide mimotope comprises amino acid sequence SWGPX₁PX₂ (SEQ IDNO:6) wherein X₁ is L, F, or analog thereof and X₂ is any amino acid oranalog thereof. In particular species of the present invention, apeptide mimotope of DON is provided comprising the amino acid sequenceSWGPFPF (SEQ ID NO:2), a peptide mimotope of DON comprising the aminoacid sequence SWGPLPF (SEQ ID NO:4), or a peptide mimotope of DONcomprising the amino acid sequence SWGPFPFGGGSC (SEQ ID NO:5). Thepeptide mimotope species are antagonistic to the inhibitory effects ofDON on in vitro protein synthesis. In a preferred embodiment, thepeptide mimotope is conjugated to a reporter for an immunological assaywherein the reporter is selected from the group consisting of alkalinephosphatase, horseradish peroxidase, or fluorescence molecule. Inanother preferred embodiment, the peptide mimotope is a part of apeptide or polypeptide. In particular, as a fusion polypeptide whereinthe polypeptide is selected from the group consisting of alkalinephosphatase and horseradish peroxidase.

The present invention further provides a nucleic acid that encodes thepeptide mimotope of DON comprising an amino acid sequence selected fromthe group consisting of SWGPLPF (SEQ ID NO:2), SWGPFPF (SEQ ID NO:4),and SWGPFPFGGGSC (SEQ ID NO:5). In particular embodiments, the nucleicacid sequence is selected from the group consisting of SEQ ID NO:1 andSEQ ID NO:3.

The present invention also provides a clone in a microorganismexpressing a peptide mimotope of DON comprising amino acid sequenceSWGPX₁PX₂ (SEQ ID NO:6) wherein X₁ is L, F, or analog thereof and X₂ isany amino acid or analog thereof. In particular species, the peptidemimotope comprises an amino acid sequence selected from the groupconsisting of SWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), andSWGPFPFGGGSC (SEQ ID NO:5). In particular, the peptide mimotopeexpressed by the clone is antagonistic to the inhibitory effects of DONon in vitro protein synthesis. For the clone expressing the peptidemimotope, the peptide mimotope is encoded by a nucleic acid in a plasmidor by a nucleic acid in a recombinant virus vector such as abacteriophage and the peptide mimotope can be expressed as an isolatedpeptide or as a part of a fusion polypeptide. Furthermore, themicroorganism containing the clone expressing the peptide mimotope canbe selected from the group consisting of bacteria and yeasts.

The present invention further provides a transgenic plant containing anucleic acid that expresses a peptide mimotope of DON that binds to amonoclonal antibody against DON and is antagonistic to the inhibitoryeffects of DON on in vitro protein synthesis. In particular, the presentinvention provides a transgenic plant that expresses a peptide mimotopeof DON comprising amino acid sequence SWGPX₁PX₂ wherein X₁ is L, F, oranalog thereof and X₂ is any amino acid or analog thereof. In particularspecies, the amino acid sequence is selected from the group comprisingSWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ IDNO:5). Further, the peptide mimotope that is expressed can be as anisolated peptide or as a part of a fusion polypeptide.

The present invention also provides an improvement in a method fordetermining whether a sample contains DON which comprises providing amonoclonal antibody against the DON, reacting the monoclonal antibodywith the sample in a reaction mixture containing a labeled DON as acompetitor, and determining whether the sample contains DON, wherein theimprovement is providing as the competitor a peptide mimotope of DON. Inparticular, the peptide mimotope has amino acid sequence SWGPX₁PX₂ (SEQID NO:6) wherein X₁ is L, F, or analog thereof and X₂ is any amino acidor analog thereof. In particular species, the amino acid sequence isselected from the group consisting of SWGPFPF (SEQ ID NO:2), SWGPLPF(SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). Further, the peptidemimotope can be as an isolated peptide or as a part of a fusionpolypeptide.

The present invention also provides a method for determining whether asample contains deoxynivalenol (DON) which comprises: (a) incubating ina reaction the sample, a monoclonal antibody against the DON, and apeptide mimotope which is a competitor of the DON for the monoclonalantibody; (b) detecting in the reaction a complex consisting of the DONbound by the monoclonal antibody and a complex formed by the mimotopeand monoclonal antibody; and (c) comparing an amount of each of thecomplexes wherein a decrease in the amount of the complex comprising thepeptide mimotope indicates that the sample contains DON. In particular,the peptide mimotope comprises amino acid sequence SWGPX₁PX₂ (SEQ IDNO:6) wherein X₁ is L, F, or analog thereof and X₂ is any amino acid oranalog thereof. In particular species, the amino acid sequence isselected from the group consisting of SWGPFPF (SEQ ID NO:2), SWGPLPF(SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). In a preferred embodimentof the method, the monoclonal antibody is produced by hybridoma cellline 6F5. The method further comprises the peptide mimotope which isconjugated to an enzyme selected from the group consisting ofhorseradish peroxidase and alkaline phosphatase; the peptide mimotopeconjugated to a fluorescent reporter; and the peptide mimotope whereinthe amino acid of the peptide mimotope is conjugated to an enzymeselected from the group consisting of horseradish peroxidase andalkaline phosphatase to make a fusion protein. Alternatively, thepeptide mimotope can be part of a fusion polypeptide wherein thepolypeptide is an enzyme that is used as a reporter enzyme inimmunoassays, in particular alkaline phosphatase or horseradishperoxidase.

The present invention also provides a kit for determining whether asample contains deoxynivalenol (DON) comprising: (a) a monoclonalantibody against the DON; (b) a peptide mimotope of the DON thatcompetes with DON for binding to the monoclonal antibody; and (c)instructions for using the kit. In particular, the mimotope comprisesamino acid sequence SWGPX₁PX₂ (SEQ ID NO:6) wherein X₁ is L, F, oranalog thereof and X₂ is any amino acid or analog thereof. In particularspecies, the peptide mimotope of the kit comprises an amino acidsequence selected from the group consisting of SWGPFPF (SEQ ID NO:2),SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). In the preferredembodiment, the monoclonal antibody is produced by hybridoma cell line6F5. The kit further comprises the peptide mimotope which is conjugatedto an enzyme selected from the group consisting of horseradishperoxidase and alkaline phosphatase; the peptide mimotope conjugated toa fluorescent reporter; and a fusion protein wherein the amino acidsequence comprising the peptide mimotope is conjugated to an enzymeselected from the group consisting of horseradish peroxidase andalkaline phosphatase. Alternatively, the peptide mimotope can be part ofa fusion polypeptide wherein the polypeptide is an enzyme that is usedas a reporter enzyme in immunoassays, in particular alkaline phosphataseor horseradish peroxidase.

The present invention further provides a method for making a plantresistant to deoxynivalenol (DON) comprising introducing into the plantplant's genome a nucleic acid that encodes a peptide mimotope, whichbinds to a monoclonal antibody against DON and is antagonistic to theinhibitory effects of DON on in vitro protein synthesis, which isoperably linked to a transcription promoter. In particular, the peptidemimotope comprises amino acid sequence SWGPX₁PX₂ (SEQ ID NO:6) whereinX₁ is L, F, or analog thereof and X₂ is any amino acid or analogthereof. In a particular species, the peptide mimotope comprises anamino acid sequence selected from the group consisting of SWGPFPF (SEQID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). Thepeptide mimotope can be expressed as an isolated peptide or as a part ofa fusion polypeptide.

The present invention also provides a method for treating an organismexposed to deoxynivalenol (DON) comprising treating the organism with apeptide mimotope of DON which is antagonistic to the inhibitory effectsof DON on in vitro protein synthesis. In particular, the peptidemimotope comprises amino acid sequence SWGPX₁PX₂ (SEQ ID NO:6) whereinX₁ is L, F, or analog thereof and X₂ is any amino acid or analogthereof. In a particular species, the peptide mimotope comprises anamino acid sequence selected from the group consisting of SWGPFPF (SEQID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). Thetreatment may be given orally, topically, or intravenously. Furthermore,the treatment can comprise a peptide mimotope of DON, which is a vaccinethat elicits antibodies against DON. The vaccine can be administeredeither as a therapeutic treatment to an animal or person displayingsymptoms of exposure to DON or as a prophylactic treatment to preventsymptoms caused by a subsequent exposure to DON. The peptide mimotopecan be the isolated peptide or as a part of a fusion polypeptide.

OBJECTS

It is therefore an object of the present invention to provide a peptidemimotope of deoxynivalenol (DON) for use in immunological assays fordetecting DON in a sample.

It is also an object of the present to provide a transgenic plant whichis resistant to the affects of DON.

It is a further object of the present invention to provide a method fortreating an animal or person exposed to DON.

DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a two-dimensional representation of DON. The asteriskindicates the site of conjugation of the carrier protein, e.g., BSA, toDON.

FIG. 1B shows a two-dimensional representation of nivalenol, an analogof DON whose three-dimensional structure is known.

FIG. 1C shows a three-dimensional structural model of the DON peptidemimotope. The white spheres represent oxygen atoms, the white cylindersrepresent nitrogen atoms, and the grey cylinders represent carbon atoms.

FIG. 1D shows a three-dimensional view of the crystallographic structureof nivalenol (CSD entry: DUTJOR10). The white spheres represent oxygenatoms, the white cylinders represent nitrogen atoms, and the greycylinders represent carbon atoms.

FIG. 1E shows a stereo view of the optimal PowerFit superposition of theknown nivalenol structure and the DON peptide mimotope structure.Nivalenol aligns with the peptide model main-chain atoms from residues 2to 5 (TrpGlyProPhe or WGPF) and partially overlaps the side chains ofTrp-2 and Pro-4. The white spheres represent oxygen atoms, the whitecylinders represent nitrogen atoms, and the grey cylinders representcarbon atoms.

FIG. 2 shows the competition between phage-displayed peptide mimotopesand DON for binding to immobilized mAB 6F5 in a CD-ELISA. Variousconcentrations of free DON competed with equal volumes ofphage-displayed peptides (at a constant concentration) for binding toimmobilized mAB 6F5. Bound phage peptide was detected withHRP-conjugated sheep anti-M13 IgG and then measured by absorbance.DON-HRP was included as a positive control.

FIG. 3A shows synthetic peptide C430 and DON competing with DON-HRP forbinding to immobilized mAB 6F5.

FIG. 3B shows synthetic peptide C430 and DON competing with C430-HRP forbinding to immobilized mAB 6F5.

FIG. 4 shows a CD-ELISA performed with DONPEP-AP fusion protein. Bindingof the DONPEP-AP fusion protein to immobilized mAB 6F5 was inhibited byfree DON. Competition of free DON with DON-HRP was used as a control.

FIG. 5 shows the use of C430 HRP and DONPEP-AP in a DON immunoassay(CD-ELISA) performed with wheat extract spiked with DON. Immulon-4microtiter wells were coated with mAB 6F5 and DON-HRP was used as apositive control.

FIG. 6 shows the specificity of antibody obtained fromC430-BSA-immunized mice. DON and C430 at various concentrations wereused to inhibit the binding of antisera to immobilized DONPEP.2 phage.Bound mouse antibodies were detected with HRP-conjugated goat anti-mouseIgG, and the amounts of these antibodies were measured by absorbance.

FIG. 7 shows the effects of DON (3.4 μM) and synthetic peptide C430 (3.4μM) on protein synthesis in vitro with rabbit reticulocyte lysate. Thetranslation template was γ-globulin mRNA. The plus and minus signsindicate which reagents were added.

DESCRIPTION OF PREFERRED EMBODIMENTS

All patents, patent applications, and literature references cited inthis specification are hereby incorporated herein by reference in theirentirety. In case of conflict, the present description, includingdefinitions, will control.

To promote a better understanding of the present invention, thefollowing terms are defined.

The term “mimotope” means a molecule which has a conformation that has atopology equivalent to the epitope of which it is a mimic. The mimotopebinds to the same antigen-binding region of an antibody which bindsimmunospecifically to a desired antigen. Generally, a mimotope willelicit an immunological response in a host that is reactive to theantigen to which it is a mimic.

The term “mimetic” is a related mimotope which means a molecule whichcompetes with the antigen for binding to the antibody but which does notelicit an antibody in a host that is reactive against the antigen. Thepresent invention is a mimetic.

The term “monoclonal antibody” as used herein refers to antibodiesproduced by a single line of hybridoma cells all directed towards oneepitope on a particular antigen. A hybridoma is a clonal cell line thatconsists of hybrid cells formed by the fusion of a myeloma cell and aspecific antibody-forming cell. In general, monoclonal antibodies are ofmouse origin; however, monoclonal antibody also refers to a clonalpopulation of an antibody made against a particular antigen or epitopeof an antigen produced by phage display technology or method that isequivalent to phage display or hybrid cells of non-mouse origin.

The term “antigen” as used herein refers to a substance which stimulatesproduction of antibody or sensitized cells during an immune response. Anantigen consists of one or more epitopes, each epitope of which iscapable of causing the production of an antibody against the particularepitope.

The term “epitope” as used herein refers to an immunogenic region of anantigen which is recognized by a particular antibody molecule. Ingeneral, an antigen will possess one or more epitopes, each capable ofbinding an antibody that recognizes the particular epitope.

Amino acids are represented herein by the single letter or triplet codewherein alanine is A or Ala, arginine is R or Arg, asparagine is N orAsn, aspartic acid is D or Asp, cysteine is C or Cys, glutamine is Q orGln, glutamic acid is E or Glu, glycine is G or Gly, histidine is H orHis, isoleucine is I or Ile, leucine is L or Leu, lysine is K or Lys,methionine is M or Met, phenylalanine is F or Phe, proline is P or Pro,serine is S or Ser, threonine is T or Thr, tryptophan is W or Trp,tyrosine is Y or Tyr, and valine is V or Val.

The nucleotides are represented herein by A for adenosine, G forguanosine, C for cytosine, and T for thymidine.

The present invention provides peptide mimotopes of the non-peptidetoxin, deoxynivalenol (DON). DON has the two-dimensional structure shownin FIG. 1A. The peptide mimotopes have a topological structure thatmimics the three-dimensional structure of DON. As shown in FIG. 1E, thethree-dimensional structure of nivalenol, an analog of DON which has thetwo-dimensional structure shown in FIG. 1B, which can be aligned alongthe peptide mimotope main chain atoms from amino acid residue 2 to 5,i.e., TrpGlyPro (WGP). The close structural alignment is sufficient toenable the peptide mimotopes to be recognized by an anti-DON monoclonalantibody, mAB 6F5, and effectively compete with DON for binding to theanti-DON monoclonal antibody (FIGS. 2-5).

Monoclonal antibody mAB 6F5 from hybridoma cell line mAB 6F5 wasprepared as described in Casale et al., J. Agric. Food Chem. 36: 663-668(1988). Monoclonal antibody mAB 6F5 is available from Michigan StateUniversity and is commercially available from Neogen Corporation, 620Lesher Place, Lansing, Mich. 48912. Alternatively, hybridomas thatproduce mAB against DON can be prepared as taught in Casale et al., J.Agric. Food Chem. 36: 663-668 (1988). Hybridoma clones that producemonoclonal antibodies against DON are then screened with a labeledpeptide mimitope which enables those hybridoma clones that react againstthe peptide mimotope to be identified.

The toxic effect of DON is caused by its binding to a particular site onthe 60S ribosome, which inhibits 60S ribosome function, therebypreventing protein synthesis. Competition experiments between DON andthe peptide mimotopes indicate that the peptide mimotopes bind to thesame site on the 60S ribosome as DON (FIG. 7); however, in contrast towhen DON is bound, the peptide mimotopes do not inhibit the function ofthe 60S ribosome. Thus, the present invention is both a mimic of the DONepitope that is recognized by the anti-DON monoclonal antibody and acompetitor that binds to the same site on the 60S ribosome as DON, butwithout DON's inhibitory effect. Since there are few examples ofmimotopes of non-proteinaceous chemicals other than biotin orcarbohydrates, it was uncertain whether a peptide sequence could befound that would mimic DON. Therefore, it was unexpected that thepeptide mimotopes which bind to the monoclonal antibody against DONwould be antagonistic to the toxic effects of DON on protein synthesisindicating it was a mimetic.

In general, the present invention provides peptide mimotopes that havean amino acid sequence of SWGPX₁PX₂ (SEQ ID NO:6) wherein the amino acidsequence mimics the DON epitope that is bound by anti-DON monoclonalantibody mAB 6F5 and wherein X₁ and X₂ is each any amino acid or analogthereof, preferably wherein X₁ is an amino acid or analog thereof whichhas a side chain that is a hydrogen or alkyl, most preferably, whereinX₁ is L, F, or analog thereof. The SWGP is the portion of the peptidethat can be aligned with the structure of nivalenol and mimics thenivalenol structure. In particular, the present invention provides apeptide mimotope that has the amino acid sequence selected from thegroup consisting of SWGPFPF (SEQ ID NO:2), which is peptide DONPEP.2;SWGPLPF (SEQ ID NO:4), which is peptide DONPEP.12; and, SWGPFPFGGGSC(SEQ ID NO:5), which is peptide C430 comprising the DONPEP.2 amino acidsequence coupled to amino acid sequence GGGSC. Each of theaforementioned peptide mimotopes are artificial peptide sequences thatare not naturally produced in nature. The peptide mimotopes can be madein vitro using any one of the peptide synthesis methods which are wellknown in the art, e.g., Fmoc peptide synthesis chemistry. For particularapplications it is desirable to produce the peptide mimotope in vivo;therefore, the peptide DONPEP.2 is encoded by the nucleic acid sequence5-AGTTGGGGTCCTTTTCCGTTT-3 (SEQ ID NO:l) and peptide DONPEP.12 is encodedby nucleic acid sequence 5′-TCTTGGGGTCCGCTTCCTTTT-3′ (SEQ ID NO:3).Producing the peptide mimotope in vivo is particularly desirable whenthe peptide mimotope is to be expressed as a part of a fusion peptide orpolypeptide. For example, the phage clones disclosed herein express achimeric polypeptide that contains the peptide mimotope amino acidsequence within and covalently linked to the amino acid sequence for theminor coat protein of phage M13, and DONPEP-AP disclosed herein is achimeric polypeptide wherein the peptide mimotope amino acid sequence isbetween and covalently linked to the amino acid sequence for the minorcoat protein signal sequence and the amino acid sequence for alkalinephosphatase. In general, chimeric fusion polypeptides, particularlylarge fusion polypeptides, are more economically produced in vivo thanin vitro.

The peptide mimotope can be synthetically produced by chemical synthesismethods which are well known in the art, either as an isolated peptideor as a part of another peptide or polypeptide. Alternatively, thepeptide mimotope can be produced in a microorganism which produces thepeptide mimotope which is then isolated and if desired, furtherpurified. The isolated or purified peptide mimotope can be used as acontrol or competitor in immunoassays for detecting DON in food samples,or because it competes with DON for binding to the 60S ribosome, theisolated or purified peptide mimotope can be used in therapies fortreating animals or humans exposed to DON. Thus, the peptide mimotopecan be produced in microorganisms such as bacteria, yeast, or fungi; ina eukaryote cells such as a mammalian or an insect cells; or, in arecombinant virus vector such as adenovirus, poxvirus, herpesvirus,Simliki forest virus, baculovirus, bacteriophage, sindbis virus, orsendai virus. Suitable bacteria for producing the peptide mimotopeinclude Escherichia coli, Bacillus subtilis, or any other bacterium thatis capable of expressing peptides such as the peptide mimotope. Suitableyeast types for expressing the peptide mimotope include, but is notlimited to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida,or any other yeast capable of expressing peptides. Methods for using theaforementioned bacteria, recombinant virus vectors, eukaryote cells toproduce peptides are well known in the art.

To produce the peptide mimotope, the nucleic acid encoding the peptidemimotope is in a plasmid and the nucleic acid is operably linked to apromoter which effects the expression of the peptide mimotope in amicroorganism. Suitable promoters include, but are not limited to, T7phage promoter, T3 phage promoter, β-galactosidase promoter, and the Sp6phage promoter. Expression of the peptide mimotope in a microorganismenables the peptide mimotope to be produced using fermentationtechnologies which are used commercially for producing large quantitiesof peptides. Methods for isolating and purifying peptides are well knownin the art and include methods such as gel filtration, affinitychromatography, ion exchange chromatography, or centrifugation.

To facilitate isolation of the peptide mimotope, a fusion polypeptide ismade wherein the peptide mimotope is translationally fused (covalentlylinked) to a heterologous polypeptide which enables isolation byaffinity chromatography. Preferably, a fusion polypeptide is made usingone of the expression systems infra. For example, the nucleic acidsequence encoding the peptide mimotope is linked at either the 5′ end or3′ end to a nucleic acid encoding a heterologous polypeptide. Thenucleic acids are covalently linked in the proper codon reading frame toenable production of a fusion polypeptide wherein the amino and/orcarboxyl terminus of the peptide mimotope is translationally fused tothe heterologous polypeptide which allows for the simplified recovery ofthe fusion polypeptide. The fusion polypeptide can also prevent themimotope polypeptide from being degraded during purification. While thefusion polypeptide is efficacious, as shown by the results herein forthe phage clones or DONPEP-AP, in some instances it can be desirable toremove the heterologous polypeptide after purification. Therefore, it isalso contemplated that the fusion polypeptide comprise a cleavage siteat the junction between the peptide mimotope and the heterologouspolypeptide. The cleavage site consists of an amino acid sequence thatis cleaved with an enzyme specific for the amino acid sequence at thesite. Cleavage sites that are contemplated include, but are not limitedto, the enterokinase cleavage site which is cleaved by enterokinase, thefactor Xa cleavage site which is cleaved by factor Xa, and the GENENASEcleavage site which is cleaved by GENENASE (GENENASE is a trademark ofNew England Biolabs, Beverly, Mass.). The following are methods forproducing the peptide mimotope as a fusion polypeptide or as an isolatedpeptide mimotope free of the heterologous polypeptide.

An example of a procaryote expression system for producing the peptidemimotope is the Glutathione S-transferase (GST) Gene Fusion Systemavailable from Amersham Pharmacia Biotech, Piscataway, N.J., which usesthe pGEX-4T-1 expression vector plasmid. The nucleic acid encoding thepeptide mimotope is fused in the proper codon reading frame with thenucleic acid encoding the GST polypeptide. The GST polypeptide allowsthe rapid purification of the fusion polypeptide using glutathioneSepharose 4B affinity chromatography. After purification, the GST can beremoved by cleavage with a site-specific protease such as thrombin orfactor Xa to produce the mimotope free of the GST polypeptide. Thepeptide mimotope free of the GST polypeptide is produced by a secondround of glutathione Sepharose 4B affinity chromatography.

Another method for producing the peptide mimotope is a method whichlinks in-frame the nucleic acid encoding the peptide mimotope to anucleic acid encoding polyhistidine, preferably encoding six histidineresidues, to produce a peptide mimotope-polyhistidine fusionpolypeptide. The polyhistidine allows purification of the fusionpolypeptide by metal affinity chromatography, preferably nickel affinitychromatography. To produce the peptide mimotope free of thepolyhistidine, a cleavage site such as an enterokinase cleavage site isfused in the proper reading frame between the codons encoding thepolyhistidine and the codons encoding the peptide mimotope. Thus, thepeptide mimotope free of the polyhistidine is made by removing thepolyhistidine by cleavage with enterokinase. A second round of metalaffinity chromatography which binds the free polyhistidine results inthe peptide mimotope free of the polyhistidine. The Xpress System,available from Invitrogen, Carlsbad, Calif., is an example of acommercial kit which is available for making and then isolatingpolyhistidine fusion polypeptides.

In a method further still, the pMAL Fusion and Purification Systemavailable from New England Biolabs can be used to make a fusionpolypeptide wherein a maltose binding protein is fused to the peptidemimotope. The maltose binding protein facilitates isolation of thefusion polypeptide by amylose affinity chromatography. The maltosebinding protein can be linked to the peptide mimotope by one of theabove mentioned cleavage sites which enables the peptide mimotope to bemade free of the maltose binding protein.

It is particularly desirable that the peptide mimotope be a part ofanother peptide or polypeptide, particularly an enzyme which is used asa reporter in immunological assays. Such reporter enzymes include, butare not limited to, alkaline phosphatase or horseradish peroxidase. Asshown herein by DONPEP-AP, the peptide mimotope was fused with alkalinephosphatase, a reporter enzyme commonly used in immunological assays. Asshown in FIG. 5, the DONPEP-AP was useful as a DON competitor inimmunological assays for detecting DON in wheat extracts. DONPEP-AP canbe used in other immunological assays for determining whether a corn,grain, or mixed feed sample is contaminated with DON. The peptidemimotope can also be fused to other heterologous polypeptides whichfacilitate isolation or handling of the peptide mimotope inimmunological assays. An example of such a heterologous polypeptideincludes, but is not limited to, the minor coat protein g3p offilamentous phage M13. Thus, the heterologous polypeptides that can beused to make fusion polypeptides include, but is not limited to, theminor coat protein g3p of filamentous phage M13, which was used to makephage clones DONPEP.1, DONPEP.2, DONPEP.3, DONPEP.4 and DONPEP.12containing the mimotope peptide sequence therein, and alkalinephosphatase, which was used to make DONPEP-AP. Preferably, the fusionpolypeptide is produced in a recombinant bacterium or eukaryoteexpression vector as disclosed supra. for producing the peptidemimotope.

Further, the mimotope peptide, either by itself or as part of a fusionpolypeptide, can be chemically conjugated to a carrier protein. Carrierproteins include, but are not limited to, bovine serum albumen (BSA),and reporter enzymes which include, but are not limited to, horseradishperoxidase or alkaline phosphatase. Further, the peptide mimotope orfusion peptide comprising the peptide mimotope can be chemicallyconjugated to fluorescence reporter molecules which include, but are notlimited to, fluorescein or R-phycoerythrin. Methods for conjugatingcarrier proteins, enzymes, and fluorescence reporter molecules topeptides and fusion polypeptides are well known in the art.

The peptide mimotopes, either alone, conjugated to a carrier protein orfluorescent reporter molecule, or as a component of a fusion polypeptideare useful as standard and conjugates in immunoassays such as ELISAs andRIAs, which are used to determine whether a food sample is contaminatedwith DON. Currently, in such immunoassays, DON, which is toxic to theuser, is used as a control or as a competitor. The immunoassays relyupon detection techniques which require DON to be conjugated to acarrier protein or reporter enzyme. Conjugating DON to a carrier proteinor reporter enzyme has been difficult and the conjugation methods whichare used require chemicals that are toxic to the user. Therefore,because the peptide mimotopes are non-toxic, the peptide mimotopesprovide a significant advantage over DON. In particular, they arenon-toxic to the user when used as a control or competitor inimmunoassays, they are easier to conjugate to reporter enzymes than DONand conjugation does not require toxic chemicals, and unlike DON, theycan be genetically fused to various reporter enzymes and produced byfermentation or other methods in large quantities thereby significantlyreducing the costs associated with providing immunoassays for detectingDON. Thus, the immunoassays of the present invention use either thepeptide mimotopes alone or the peptide mimotopes conjugated to a carrierprotein or enzyme, or genetically fused to a reporter enzyme such asalkaline phosphatase or horseradish peroxidase, or conjugated to areporter fluorescence molecule.

In general, the immunoassays are performed using an enzyme-linkedimmunosorbent assay (ELISA) embodiment and can be either a competitivedirect ELISA (CD-ELISA) or competitive indirect ELISA (CI-ELISA). Toperform a CD-ELISA, a microtiter plate is provided containing aplurality of wells wherein a first well or series of wells contains amonoclonal antibody against DON immobilized to the surface therein,preferably the monoclonal antibody is mAB 6F5. To prevent non-specificbinding in the subsequent steps, it is preferable that the wells betreated with a blocking agent such as a 10% solution of non-fat milk.Next, a limiting dilution series of an aliquot of the sample are mixedwith an equal volume of an appropriate dilution of the peptide mimotopeand the mixture added to the wells containing the bound monoclonalantibody. Preferably, the mimotope peptide is conjugated to a carrierprotein or is part of a fusion polypeptide. The DON in the sample andthe peptide mimotope compete for binding to the monoclonal antibody. TheELISA is incubated for a time sufficient for monoclonal antibody-DONcomplexes to form. In general, an incubation time of about an hour at atemperature between about room temperature and 37° C. Afterwards, thewells are washed to remove any unbound material. The wells are thenincubated with a labeled antibody or labeled monoclonal antibody thatbinds to the carrier protein or fusion polypeptide to form a complexwhich can be detected when the labeled monoclonal or polyclonal antibodyis conjugated to a reporter ligand such as horseradish-peroxidase oralkaline phosphatase. Preferably, the incubation is for about an hour ata temperature between about room temperature and 37° C. A detectablesignal from the reporter indicates the sample does not contain DONwhereas an absence of a signal indicates that the sample contains DONwhich had bound all of the monoclonal antibody, thereby preventing thepeptide mimotope from binding the monoclonal antibody immobilized in thewells. Alternatively, the second monoclonal or polyclonal antibody canbe conjugated to reporter ligands such as a fluorescing ligand, biotin,colored latex, colloidal gold magnetic beads, radioisotopes or the like.When the fusion polypeptide comprises a reporter enzyme such as alkalinephosphatase, the antibody-mimotope peptide complex can be detecteddirectly without the need for a labeled antibody. In either case,detection is by methods well known in the art for detecting theparticular reporter ligand.

To perform a CI-ELISA, a microtiter plate is provided containing aplurality of wells wherein a first well or series of wells contains thepeptide mimotope, the peptide mimotope conjugated to a carrier protein,or fusion polypeptide comprising the peptide mimotope is immobilized tothe surface therein. To prevent non-specific binding in the subsequentsteps, it is preferable that the wells be treated with a blocking agentsuch as a 10% solution of non-fat milk. Next, a limiting dilution seriesof an aliquot of the sample are added to the wells containing the boundpeptide mimotopes along with a constant amount of a monoclonal antibody,preferably the mAB 6F5 monoclonal antibody. The DON in the sample andthe peptide mimotope bound to the well surfaces compete for binding tothe monoclonal antibody. The ELISA is incubated for a time sufficientfor antibody-DON complexes to form. In general, an incubation time ofabout an hour at a temperature between about room temperature and 37° C.Afterwards, the wells are washed to remove any unbound material. Theamount of monoclonal antibody that is bound to the immobilized mimotopepeptides in the well is determined by incubating the wells with alabeled antibody or labeled monoclonal antibody that binds to themonoclonal antibody to form a complex that can be detected when thelabeled monoclonal or polyclonal antibody is conjugated to a reporterligand such as horseradish-peroxidase or alkaline phosphatase.Preferably for about an hour at a temperature between about roomtemperature and 37° C. A detectable signal from the reporter indicatesthe sample does not contain DON whereas an absence of a signal indicatesthat the sample contains DON which had bound all of the monoclonalantibody, thereby preventing the monoclonal antibody from binding thepeptide mimotope immobilized in the wells. The intensity of the signalprovides an estimate of the relative concentration of DON in the sample.Alternatively, the second monoclonal or polyclonal antibody can beconjugated to reporter ligands such as a fluorescing ligand, biotin,colored latex, colloidal gold magnetic beads, radioisotopes or the like.In an alternative further, the monoclonal antibody can be labeled with areporter in which case the bound monoclonal antibody can be detecteddirectly without the need for a labeled antibody. In either case,detection is by methods well known in the art for detecting theparticular reporter ligand.

Instead of an ELISA, the peptide mimotopes can be used in a radioimmunoassay (RIA) for detecting DON in a sample. The RIA procedureinvolves incubation of a monoclonal antibody against the DON, preferablymAB 6F5, simultaneously with a solution of unknown sample or knownstandard, and a constant amount of radioactively labeled peptidemimotope or fusion polypeptide. After separation of the free peptidemimotope or fusion polypeptide from bound peptide mimotope or fusionpolypeptide, the radioactivity in the respective fractions isdetermined. The concentration of DON in the unknown sample is determinedby comparing results to a standard curve. Several known methods,including the ammonium sulfate precipitation method, double antibodytechnique, solid phase RIA method in which immunoglobulin G (IgG) isconjugated to CNBr-activated SEPHAROSE gel (Pharmacia Biotech,Piscataway, N.J.), a dextran-coated charcoal Column and albumen-coatedcharcoal, is used for the separation of free from bound peptide mimotopeor fusion polypeptide in the RIA. Radioactivity is determined in aliquid scintillation counter in 5 ml of AQUASOL (a product of NewEngland Nuclear Corp., Boston, Mass.) for aqueous solutions.

In a further embodiment of the present invention, the peptide mimotopeor fusion polypeptide is coupled to an energy donor fluorophore and themonoclonal antibody is coupled to an energy acceptor or quencherfluorophore. The quencher fluorophore is attached to a monoclonalantibody against DON, preferably mAB 6F5, such that when the peptidemimotope or fusion polypeptide binds the antibody, the quencher andreporter dye are in close proximity and the reporter dye is preventedfrom fluorescing. Therefore, when a sample does not contain DON, all ofthe peptide mimotope or fusion polypeptide is bound by the monoclonalantibody. Since the quencher and reporter dyes are in close proximity,the quencher prevents the reporter dye from fluorescing. However, when asample contains DON, the DON competes with the peptide mimotope offusion polypeptide for the antibody, which results in some peptidemimotope or fusion polypeptide molecules remaining unbound. Becausethese unbound peptide mimotope or fusion polypeptide molecules are nolonger in close proximity to the quencher on the antibody, the reporterdye on these molecules will fluoresce. The intensity of fluorescence isdirectly proportional to the amount of DON in the sample. Fluorescencecan be detected with an ABA Prism Sequence Detector or TAQMAN LS-50B PCRDetection System (both available from Perkin-Elmer Applied Biosystems),or other detector that is used to detect fluorescence. Alternatively,the peptide mimotope or fusion polypeptide is coupled to an energyacceptor fluorophore and the monoclonal antibody is coupled to theenergy transfer fluorophore. The result would be the same. Preferably,the fluorophore is selected from the group consisting of fluorescein,5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine(R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA),6-carboxyl-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo) benzoic acid(DABCYL), tetrachloro-6-carboxyl-fluorescein (TET), VIC, and5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Thisembodiment can be performed in a small reaction volume, does not need torely on microtiter plates, and enables the assay results to be knowninstantaneously.

Since the ability to test samples in the field for DON contamination isvery important, the method of the present invention further includesrapid immunodiffusion based methods and apparatuses for detecting DON ina sample. For example, a device containing the peptide mimotope, eitheras the peptide or as a fusion polypeptide is irreversibly fixed to asolid support. A solution containing the test sample is admixed with asolution containing a monoclonal antibody against DON, preferably mAB6F5, conjugated to a reporter and a solution containing the solidsupport containing the mimotope. The admixture is then applied to aporous sheet material incorporating a chromogen and a substrate for thereporter, while keeping the support containing the mimotope out ofcontact with the porous sheet material. If the sample contains DON, theDON complexes with the antibody conjugate, which causes a color reactionon the porous sheet material. If the sample does not contain DON, theantibody is completely bound by the peptide mimotope or fusionpolypeptide on the solid support. Since there is no antibody insolution, there is no color reaction. This method is disclosed in U.S.Pat. No. 5,846,745 to Christensen et al. The present invention can beused solid phase immunodiffusion assays such as those disclosed in U.S.Pat. No. 5,169,789 to Berstein. The above methods are provided asexamples thus, other rapid immunoassays which are well known in the artare also within the scope of the present invention.

Thus, the present invention can be provided as a kit that comprises anyone of the methods described above or in U.S. Pat. No. 5,620,845 toGould et al., U.S. Pat. No. 5,559,041 to Kang et al., U.S. Pat. No.5,656,448 to Kang et al., U.S. Pat. No. 5,728,587 to Kang et al., U.S.Pat. No. 5,695,928 to Stewart et al., U.S. Pat. No. 5,169,789 toBernstein et al. U.S. Pat. No. 4,486,530 to David et al., and U.S. Pat.No. 4,786,589 to Rounds et al. While the aforementioned discloseparticular rapid immunodiffusion methods, the present invention is notto be construed to be limited to the aforementioned. It is within thescope of the present invention to embrace derivations and modificationsof the aforementioned.

When the peptide mimotopes are conjugated to an appropriate compound orchemical that facilitates entry of the peptide mimotopes into the cellof the host, the peptide mimotopes can be used as a treatment forplants, animals or people exposed to DON. Alternatively, the peptidemimotope can be a part of a peptide or polypeptide, i.e., fusionpolypeptide or polypeptide, that facilitates entry of the peptidemimotope into the cell. The peptide mimotope in any of theaforementioned forms can be administered either topically, orally, or byinjection.

The present invention further includes transgenic plants that expressthe peptide mimotope, either as an isolated peptide or as a part of afusion peptide or polypeptide which renders the plant resistant to theeffects of DON. For example, the Fusarium fungi produce a broad range ofplant diseases such as seedling and head blight on small grains such aswheat and rye, ear and stalk blight on corn (maize), stem rot ofcarnation, and seedling blight and root rot of a number of other plantspecies, including, beans, clover, peanuts, and tomato. Therefore, thepresent invention provides a method for making transgenic plantsresistant to DON produced by Fusarium fungi. Since the peptide mimotopecompetes with DON for binding to the 60S ribosome but does not haveDON's inhibitory effect, transgenic plants expressing the presentinvention are resistant to the effects of DON. Transgenic plants thatexpress the peptide mimotope, either as an isolated peptide or as partof a fusion peptide or polypeptide, include, but are not limited to,wheat, rye, corn, carnation, beans, clover, and tomato.

Therefore, the transgenic plant of the present invention comprises anucleic acid that encodes a peptide mimotope which has the amino acidsequence SWGPX₁PX₂ (SEQ ID NO:6) wherein X₁ and X₂ is each any aminoacid or analog thereof, preferably wherein X₁ is an amino acid or analogthereof which has a side chain that is a hydrogen or alkyl, mostpreferably, wherein X₁ is L, F, or analog thereof. In particular, theamino acid sequence which is set forth in SEQ ID NO:2, SEQ ID NO:4, orSEQ ID NO:5. In a preferred embodiment, the peptide mimotope is encodedby a nucleic acid comprising the nucleotide sequence selected from thegroup consisting of SEQ ID NO:1 and SEQ ID NO:3. As disclosed supra.,the nucleic sequence encoding the peptide mimotope can be covalentlylinked in frame with a sequence encoding a heterologous peptide orpolypeptide.

Expression of the peptide mimotope, or peptide or polypeptide comprisingthe peptide mimotope, in the plant cell requires the nucleic acidencoding the mimotope be operably linked to a transcription promoterthat is functional in plant cells. Examples of promoters which areuseful are viral promoters such as the cauliflower mosaic virus 35Spromoter, heat shock protein promoters such as the HSP70 promoter, lightinduced promoters such as the ST-Ls1 or the rubisco small subunitpromoter, stress response promoters such as the PR promoter, theAgrobacterium tumefaciens nos promoter, and various organ, root, tuber,and leaf specific promoters. The DRE promoter element that is inducibleunder stress is an example of a plant promoter that responds toenvironmental conditions (Yamaguchi-Shinozaki et al., Plant Cell 6:251-264 (1994)). The nucleic acid encoding the peptide mimotope ispreferably operably linked at the 3′ end to a transcription terminationsignal. An example of such a sequence is the transcription terminationsignal of the octopine synthase gene.

There are many methods known in the art for transforming a plant cellwith heterologous nucleic acids. Common methods include transformationwith T-DNA containing the DNA of interest and using Agrobacteriumtumefaciens as the means for transformation or with Ti or Ri plasmidsusing the bacterium A. rhizogenes as the means for transformation. Asuitable plasmid for transformations is the pART27/7 plasmid vectorisolated from Agrobacterium tumefaciens. Other methods for transforminga plant cell include cell fusion, electroporation, biolistic orconventional injection.

Agrobacterium related methods require special plasmid vectors such asintermediate or binary vectors. Intermediate vectors require integrationinto Ti or Ri plasmids by homologous recombination into the regioncontaining the T-DNA. The intermediate vector is transferred into theAgrobacterium by means of conjugation in the presence of a helperplasmid. The transformed Agrobacterium is then used to transform thecell. The preferred method for transforming Agrobacterium is usingplasmids of the binary type. Binary vectors replicate both inEscherichia coli and Agrobacterium. Therefore, these vectors containingthe desired DNA can be constructed using conventional molecular biologytechniques and the recombinant plasmid directly transferred toAgrobacterium. Binary vectors usually contain a marker gene and apolylinker for inserting the desired DNA flanked by the left and rightT-DNA border regions. Both the intermediate and binary vectors containthe vir region which is necessary for transfer of the T-DNA into theplant cell.

Transformation of plant cells with transformed Agrobacterium is byco-cultivation of the cells with the transformed Agrobacterium whichresults in transfer of the T-DNA containing the desired nucleic acidinto the plant cell. Sources for plant cells are explants which caninclude but is not limited to sections of leaves, stems, roots, segmentsof petioles, flowers and flower parts, and cotyledon tissue. Wholeplants are regenerated from the infected plant material or fromprotoplasts or suspension-cultivated cells in a suitable medium whichcan contain antibiotics or biocides (e.g., kanamycin, bleomycin,hygromycin, chloramphenicol) for selection of the transformed plantcells. The ability and efficiency of regenerating a transformed ortransgenic plant using transformed isolated cells or explants isdependent on the species of plant and the type of transformed cell.Transformation of plants can be achieved according to theAgrobacterium-mediated method disclosed in U.S. Pat. No. 5,684,238 toAusich et al and U.S. Pat. No. 5,618,988 to Hauptmann et al.

Non-Agrobacterium mediated transformation such as electroporation,injection, cell fusion, or particle bombardment do not require specialplasmids and, therefore, can use standard plasmids such as the pUCderivatives and conventional cloning techniques. For example, to makethe transgenic plants of the present invention using the Biolisticbombardment method, plant tissue is transformed using the Biolisticmethod described in U.S. Pat. No. 5,767,368 to Zhong et al. Furtherexamples of the Biolistic bombardment method are disclosed U.S. Pat. No.5,736,369 to Bowen et al.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

Identification of peptide mimotopes which bind to mycotoxin DON-specificmonoclonal antibody. Monoclonal antibody 6F5 (mAB 6F5) was used toselect for peptides that mimic the mycotoxin from a library offilamentous phages that have random 7-mer peptides on their surfaces.

A phage-display heptapeptide library containing 2×10⁹ independent clonesthat express random peptide 7-mers fused to minor coat protein g3p offilamentous coliphage M13 was purchased from New England Biolabs, Inc.Beverly, Mass. The library had sufficient complexity to contain most ifnot all of the 20⁷=1.28×⁹10 possible 7-mer sequences. Afteramplification in Escherichia coli ER2537 (purchased from New EnglandBiolabs, Inc.), the phage in the library were selected by panningelution as below.

One hundred microliters of a preparation containing mAB 6F5 (prepared asshown in Casale et al., Op. cit.) at 15 μg/ml in 0.01 Mphosphate-buffered saline (PBS) (pH 7.4) was dispensed into each well ofdisposable IMMUNO-4 microtiter strips (Dynatech Laboratories, Inc.,Chantilly, Va.). The antibody was dried overnight onto the wells in aforced-air oven at 40° C. Blank wells were coated with an equalconcentration of mouse IgG. The wells were washed 4 times by fillingeach well with 300 μl PBS and aspirating the contents. Nonspecificbinding was blocked by incubating 320 μl of 10% non-fat dry milkdissolved in PBS (10% milk-PBS) in each well for 1 hour at 37° C. andthen washing the wells four times with PBS. For panning-elutionselection, the recombinant phage display peptide library, which had beendiluted with 10% milk-PBS to about 10¹⁰ PFU/ml, was added to the wells(100 μl/well) and the wells incubated at 37° C. for 1 hour. Afterwards,the wells were washed 20 times by filling each well with about 300 μl ofPBS containing 0.1% Tween 20 (PBS-T), followed by incubation with 300 μlof PBS per well at 37° C. with shaking at 150 rpm for 1 hour. The wellswere then washed 10 times with about 300 μl PBS-T per wash. To elute thebound phage, 100 μl of DON (100 μg/ml in PBS containing 1% methanol) orPBS containing 1% methanol was added to each well, and incubated at 37°C. with shaking at 150 rpm for 1 hour. The eluted phage were collectedfrom the microtiter wells and used to infect E. coli ER2537 for phagetiter and amplification experiments. The amplified phage, whichcontained about 8 ηg/ml of DON, was used for a subsequent round ofpanning-elution selection. After four rounds of panning-elutionselection, individual plaques were picked from Luria-Bertani plates andused to infect E. coli ER2537 cells in phage production experiments.

Binding to mAB 6F5 was determined for each individual phage clone byELISA. The amounts of bound phage in mAB 6F5-coated microtiter wellswere determined by incubation with 100 μl of sheep anti-M13 horseradishperoxidase (HRP) conjugate, which had been diluted 1:5,000 in 10%milk-PBS, per well at 37° C. per hour, followed by incubation with 100μl of 3,3′,5,5′-tetramethylbenzidine substrate per well at 37° C. for 15minutes. The absorbance at 450 ηm was determined after the reaction wasstopped by adding 100 μl of 10% sulfuric acid per well.

After specific binding to mAB 6F5 was confirmed by ELISA, 10 ml ofrecombinant phage particles (10¹¹ PFU/ml in LB medium) from eachpositive phage clone was used for single-stranded DNA isolationperformed with a QIAPREP Spin M13 kit (Qiagen, Inc., Chatsworth,Calif.). The single-stranded DNA was sequenced with −28 gIII and −96gIII sequencing primers (New England Biolabs, Inc.) by using Taq cyclesequencing and dye terminator chemistry at the Michigan State UniversityDNA Sequencing Facility.

Five phage isolates were identified to bind to mAB 6F5 by ELISA. Theseisolates were designated DONPEP.1, DONPEP.2, DONPEP.3, DONPEP.4, andDONPEP.12. Four of the five isolates had the same nucleotide sequencewhich was 5-AGTTGGGGTCCTTTTCCGTTT-3 (SEQ ID NO:1), which encodes thepeptide with the amino acid sequence SWGPFPF (SEQ ID NO:2) while isolateDONPEP.12 had the nucleotide sequence 5′-TCTTGGGGTCCGCTTCCTTTT-3′ (SEQID NO:3), which encodes the peptide with the amino acid sequence SWGPLPF(SEQ ID NO:4).

EXAMPLE 2

This example demonstrates that DON mimotope peptides actually mimickedthe epitope recognized by mAB 6F5 and was not nonspecifically bound tothe surface of the antibody molecule outside the antigen binding site.Competitive ELISAs were used to show that both phage isolates encodingeither peptide competed with DON for the antigen binding site.

To perform a competitive direct ELISA (CD-ELISA) with thephage-displayed peptide, IMMUNO-4 microtiter wells were coated with mAB6F5 and blocked as described in Example 1. Various concentrations of DON(0 to 10,000 ηg/ml in 1% methanol-PBS) were mixed with equal volumes ofphage-displayed peptide (diluted 1:10 in 10% milk-PBS). The mixtureswere added to mAB 6F5-coated microtiter wells (100 μl/well), and thepreparations were incubated at 37° C. for 1 hour. Afterwards, the wellswere washed 6 times with PBS-T (about 300 μl/well) and then amounts ofbound recombinant phage were determined by incubating the preparationswith 100 μl of sheep anti-M13 HRP conjugate (diluted 1:5,000 in 10%milk-PBS) per well at 37° C. for 1 hour. The amounts of bound enzymewere determined as described in Example 1. For comparison, 50 μl ofDON-HRP per well was also mixed with 50 μl of DON at variousconcentration (0 to 10,000 ηg/ml in 1% methanol-PBS) per well, and thepreparations were incubated in mAB 6F5-coated microtiter wells at 37° C.for 1 hours. The amounts of bound enzyme were determined as above.

To perform a competitive indirect ELISA (CI-ELISA) with phage-displayedpeptide, 100 μl of phage-displayed peptide was dispensed into each wellof disposable IMMUNO-4 microtiter strips, and the peptide was dried ontothe wells in a forced air oven at 40° C. overnight. The strips werewashed and blocked as described for the CD-ELISA. Various concentrationsof DON (0 to 10,000 ηg/ml in 1% methanol-PBS), 50 μl/well, was added tothe wells, and then 50 μl of anti-DON mAB 6F5 (10 μg/ml in 10% milk-PBS)was added to each well. The wells were incubated at 37° C. for 1 hour.Then the wells were washed six times with PBS-T, and the amounts ofbound anti-DON mAB 6F5 were determined by incubation with goatarti-mouse IgG-HRP conjugate (diluted 1:2,000 with 10% milk-PBS) at 37°C. for 1 hour. The amounts of bound enzyme were determined as describedabove.

The results are shown in FIG. 2. The binding of phage clones DONPEP.2and DONPEP.12 to immobilized mAB 6F5 was competitively inhibited by freeDON. This strongly suggested that these two phage clones bind to theantigen binding site of the mAB, mimicking, in part, the structuralepitope of DON.

EXAMPLE 3

This example demonstrates that a synthetic peptide, C430, whichcomprises the amino acid sequence SWGPFP (SEQ ID NO:2), was alone wassufficient for binding to the mAB, independent of the phage structuralcontext.

C430, a DON peptide mimotope with a structurally flexible linker and acysteine residue, which has the sequence NH₂-SWGPFPFGGGSC-COOH (SEQ IDNO:5) was synthesized via N-(9-fluorenylmethoxycarbonyl) (Fmoc)chemistry at Bio-Synthesis, Inc. of Lewisville, Tex. C430 was used atvarious concentrations to compete with DON-HRP for binding to mAB 6F5 ina CD-ELISA. C430 was also conjugated to HRP with a sulfo-MBScross-linker. The procedure used for the CD-ELISA performed with theC430-HRP conjugate was the same as the procedure used for the ELISAperformed with DON-HRP described above, except that C430-HRP (diluted1:5,000 in blocking buffer) replaced the DON-HRP.

The results shown in FIG. 3A show that binding of DON-HRP to immobilizedmAB 6F5 was inhibited by free C430, and the results shown in FIG. 3Bshow that binding of C430-HRP conjugate was inhibited by free DON. Thisindicates that the peptide alone was sufficient for binding to the mAB,independent of the phage structural context. When C430 was used tocompete with DON-HRP or C430-HRP conjugate for binding to mAB 6F5, the50% inhibitory concentrations were 0.64 to 0.8 μM, whereas 3.4 μM freeDON was required to obtain 50% inhibition of DON-HRP or C430-HRPconjugate binding to the same mAB. This indicates that mAB 6F5 has ahigher affinity for C430 than for DON. In a similar CD-ELISA, none ofthe individual amino acids in C430 (at concentrations up to 34 μM)significantly inhibited binding to DON-HRP. This suggests that thesequence in C430 was important for specific binding to mAB 6F5, sinceindividual amino acids did not bind mAB 6F5 specifically (data notshown).

EXAMPLE 4

This example demonstrates that the DON mimotope peptide is structurallystable in a protein context different from the phage protein context oras a free peptide.

A 179-bp DNA fragment encoding the g3p signal peptide, DONPEP.2, and thefirst 17 amino acids of M13 phage g3p protein from the DONPEP.2 phageisolate was amplified by polymerase chain reaction (PCR) performed withPfu DNA polymerase, a sense primer,5-GCCAAGCTTAGATCTTGGAGCCTTTTTTTTGGAG-3′ (SEQ ID NO:7), and an antisenseprimer, 5′-CCGGTCGACCTGTATGGGATTTTGCTAAACAACT-3′ (SEQ ID NO:8). Aftergel purification, the amplified DNA was digested with BglII and SalI andwas cloned into BamHI-SalI-digested pLIPS (Carrier et al., J. Immunol.Methods 181: 177-186 S/(1995)), which generated plasmid pQY7 containingthe nucleic acid sequence encoding DONPEP.2 covalently linked to the 5′end of the nucleic acid sequence encoding alkaline phosphatase. Theligated product was used to transform E. coli DH11S competent cells(GIBCO BRL, Gaithersburg, Md.), which generated DH11pQY7.DONPEP-alkaline phosphatase (AP) fusion protein was produced fromDH11S/pQY7 grown in SB medium (35 g bactotrypase, 20 g bacto yeastextract, 5 g NaCl per liter) containing 100 μg/ml ampicillin and inducedby 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The periplasmicDONPEP-AP fusion protein was extracted by suspending the bacterial cells(1:5, vol:vol) in lysis buffer (50 mM Tris-HCl [pH 8.0], 20% sucrose, 10mM EDTA, 0.1 mg/ml lysozyme, 0.5 mM phenylmethylsulfonyl fluoride).After 1 hour on ice with agitation, the preparation was centrifuged at7,000×g for 30 minutes. The supernatant fraction was filtered through a0.4 μm-pore-size porous filter. The ability of the DONPEP-AP fusionpeptide to compete with DON for binding to mAB 6F5 was determined byCD-ELISA performed as previously described. Briefly, IMMUNO-4 wells werecoated with mAB 6F5 and washed as described above. Serial dilutions ofDON (0 to 10,000 ηg/ml in 0.05 M Tris-buffered saline pH 7.4 (TBS) wereeach mixed with equal volumes of DONPEP-AP fusion protein (periplasmicextract diluted 1:7 with 2% nonfat dry milk-TBS). The mixtures (100μl/well) were added to the mAB 6F5 coated microtiter wells and incubatedat 37° C. for 1 hour. After the wells were washed six times with 300 μlof TBS containing 0.1% Tween 20, the amounts of bound DONPEP-AP for eachmixture were determined by incubation with a p-nitrophenyl phosphatesubstrate solution at 37° C. for 45 minutes. The absorbance at 405 nmwas determined.

The results, which are shown in FIG. 4, show that the DONPEP-AP fusionprotein had alkaline phosphatase activity, but more importantly, theresults show that its specific binding to mAB 6F5 was similar to that ofDON-HRP. This indicates that the DON peptide mimotope sequence isstructurally stable in a different protein structural context.

EXAMPLE 5

This example demonstrates the feasibility of using DON mimotope peptidesequence as immunochemical reagents for DON immunoassays in food andfeed.

A CD-ELISA was performed as above using wheat extracts spiked with DON.As shown in FIG. 5, both the C430-HRP conjugate and DONPEP-AP fusionprotein exhibited binding to immobilized mAB 6F5 in the wheat extractwhich was similar to the binding of DON-HRP. All three HRP conjugatesproduced similar linear inhibition curves at DON concentrations rangingfrom 0.1 to 10 μg/ml in wheat extract. However, a slightly lower levelof absorbance was observed in the CD-ELISA performed with C430-HRP andDONPEP-AP in wheat extract than in PBS buffer, indicating that the wheatextract interfered with binding of the peptide mimotope to anti-DON mAB6F5 to some extent.

EXAMPLE 6

This example was performed to determine whether the DON peptide mimotopesequence can elicit an immune response in animals similar to that of DONand, therefore, be useful as an alternative antigen for DON.

Synthetic peptide C430 was conjugated to BSA with am-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS)cross-linker. The C430-BSA conjugate, at a molar ratio of peptide C430to the carrier protein BSA of about 29:1, was used to inject 7-week-oldBALB/c female mice intraperitoneally or 6-month-old New Zealand whiterabbits subcutaneously. The initial injections used for the micecontained 100 to 200 μg of C430-BSA conjugate in 200 μl ofsaline-Freund's complete adjuvant (1:1). These injections were followedat 3-week intervals by booster injections consisting of 100 to 200 μg ofconjugate in 200 μl of saline-Freund's incomplete adjuvant (1:1). Theinitial inoculum used for rabbits consisted of 1 mg of the C4430-BSAconjugate in 1 ml of saline Freund's complete adjuvant (1:1) followed bybooster injections at 4-week intervals consisting of 250 μg of conjugatein 1 ml of saline-Freund's incomplete adjuvant. The animals were bled 1week after each booster injection. The antisera were screened forspecific binding in phage-displayed DONPEP.2 coated wells by CI-ELISA asdescribed above in which C430 or DON was used as the complete adjuvant.

Both the mice and rabbits produced antibody specific to the DON peptidemimotope sequence after the second injection of C430-BSA conjugate. FIG.6 shows that after the fourth injection, the anti-serum exhibited astrong antibody response against the DON peptide mimotope sequence. Asynthetic peptide C430 concentration of 0.39 μM resulted in 50%inhibition of antiserum binding to the immobilized phage-displayedpeptide. However, binding of antisera to immobilized phage-displayedpeptide was not inhibited by free DON in solution, indicating that theantibodies in the immunized animals were not specific for DON. Thus, thepeptide mimotope sequences do not represent a true image of the DONsurface structure.

EXAMPLE 7

This example was to determine whether the DON peptide mimotope sequencehas any cytotoxic effect. It has been known that one of the effects ofDON is to cause cytotoxicity through cell apoptosis and whether thepeptide mimotope sequences were like DON and had an effect on newprotein synthesis.

Comparisons of the cytotoxic effect by the peptide mimotope sequences tothe effects by DON were performed on mouse bone marrow cells.Ten-week-old B6C3F1 mice were euthanized by cervical dislocation, andthe femurs were removed. The bone marrow cells were flushed from thefemurs by using a 1-ml syringe and a 25-gauge needle. Erythrocytes werelysed with 0.83% ammonium chloride. Cell number and viability weredetermined by trypan blue dye exclusion by using a hemacytometer. Thecells were cultured at a concentration of 10⁶ cells/ml in 96-wellflat-bottom plates in RPMI-1640 in a humidified incubator containing 5%CO₂. RPMI-1640 was supplemented with 100 units penicillin per ml, 100 μgof streptomycin per ml, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 1mM nonessential amino acids, 2 mM glutamine, and 10% fetal bovine serum.DON and synthetic peptide C430 were separately diluted in RPMI-1640. Thefinal concentrations of C430 were 0.34, 3.4. and 34 μM and the finalconcentration of DON was 3.4 μM. Controls consisted on RPMI-1640.Duplicate cultures were treated. After 18 hours of exposure to DON orC430, cell viability was determined by using an MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]conversion assay as described by Marin et al. in Toxicology 114: 67-79(1996).

As expected, DON at a concentration of 3.4 μM caused 40 to 60% celldeath after 18 hours of incubation. In contrast, synthetic peptide C430did not have any adverse effect on the viability of the bone marrowcells at any of the concentrations tested after 18 hours incubation.When combined with DON, C430 at any of the above test concentrations didnot significantly increase or decrease the cell viability caused by DON.This indicates that there was no significant synergism or antagonismbetween the DON peptide mimotope and DON with respect to bone marrowcell death.

EXAMPLE 8

This example was to determine whether the DON peptide mimotope sequencewere like DON and had an effect on new protein synthesis.

To determine the effects on protein synthesis, in vitro translationassays were performed using γ-globulin mRNA template. DON or syntheticpeptide C430 (0 or 3.4 μM in a 50-μl (final volume) reaction mixture)was added to 30 μl of a biotin translation mixture (Boehringer MannheimCorp., Indianapolis, Ind.) containing reticulocyte lysate, 10 pmol ofbiotin-lysine-tRNA^(lys), 42 μM amino acids (without lysine),spermidine, energy mixture, dithiothreitol, 83 mM potassium acetate, and83 mM magnesium acetate. The mixtures (46 μl) were incubated on ice for10 minutes, and then 4 μl of γ-globulin mRNA (0.5 μg/ml) was added. Thefinal reaction mixture (50 μl) was incubated at 30° C. for 1 hour. Thetranslated protein samples were stored at −80° C. before analysis byWestern blot.

For Western blot analysis, 3 μl aliquots of protein from the in vitrotranslation assays were boiled for 10 minutes after mixing with sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis loading buffer.The samples were then loaded into the wells of a mini SDS −10%polyacrylamide gel and electrophoresed at 80 volts for 2 hours. The invitro synthesized proteins were detected by electrotransfer to apolyvinylidene difluoride transfer membrane (DuPont NEN ResearchProducts, Boston, Mass.) followed by incubation withstrepavidin-peroxidase conjugate (Boehringer Mannheim Corp.). Boundenzyme was visualized by incubation with SUPERSIGNAL ULTRAchemiluminescent substrate (Pierce Chemical Co., Rockford, Ill.) and byexposing to Kodak XAR5 autoradiography film (Kodak Corp., Rochester,N.Y.).

As shown in FIG. 7, DON at a concentration of 3.4 μM significantlyinhibited new protein synthesis whereas 3.4 μM of C430 had no suchinhibitory effect. Unexpectedly, when 3.4 μM DON was mixed with anequimolar amount of C430, new protein synthesis was not inhibited. Thisindicates that at least in vitro, C430 has an antagonistic to theinhibitory effect of DON on protein synthesis.

EXAMPLE 9

This example provides a structural model of the DON peptide mimotopesequence and compares the structural model with the structure of DON.

To do homologous sequence searching and peptide modeling, the computerprogram SEQUERY (Collawn et al., EMBO J. 10: 3247-3252 (1991); Craig etal., J. Molec. Biol. 281: 183-201 (1998)) was used to search for aminoacid sequences similar to the DONPEP.2 sequence (SWGPFPF) in a databaseof nonhomologous protein structures derived from the ≦25% identity setof the Protein Data Bank (PDB) Select list. SEQUERY identified alloccurrences of protein sequences in the PDB that matched thetetrapeptidyl fragments of DONPEP.2. The following conserved amino acidresidue substitutions were allowed during the sequence search: S for T,W for F, P for ST, and F for ST. The resulting sequence analogsconsisted of tetrapeptides that matched a portion of DONPEP.2 and hadknown three-dimensional structures which were available in theBrookhaven PDB. The secondary structures of these sequence analogs weresubsequently analyzed using the Superpositional Structural Assignmentcomputer program (Craig et al., J. Molec. Biol. 281: 183-201 (1998)).This program superimposed the sequence analogs onto a set of regularsecondary structure templates and assigned each sequence analog to thestructural category, e.g., α-helix, β-strand, reverse turn, whichmatched most closely within a 1.0-Å main-chain root-mean-squarepositional deviation (RSMD); if no template matched a 1.0-Å RSMD, thestructure of the sequence analog was considered irregular. The sequenceanalogs that were found to be irregular were analyzed visually by usingmolecular graphics, so that slightly irregular structures, e.g., a bentα-helical turn, could be assigned to the most appropriate structuralcategory.

A three-dimensional structural model of DONPEP.2 was created based onthe structural sequence analogs by using the molecular modeling softwareINSIGHT II (available from Molecular Simulations Inc., San Diego,Calif.) and a Silicon Graphics INDIGO² EXTREME computer (Mountain View,Calif.). The overlapping amino acid residues of the sequence analogswere superimposed, and a consensus structure was created, whichconsisted of the peptide main-chain atoms and proline side chains. Sidechains for the nonproline residues were added to the model by using therotamer library of INSIGHT II. Final side chain positions were selectedbased on consensus between the sequence analogs and the absence ofsteric overlap. Interactions within the model were then optimized byusing 100 steps of backbone-restrained steepest descent energyminimization with the cff91 force filed of the Discover 3 module ofINSIGHT II. The stereochemical quality of the model was validated withPROCHECK (Laskowski et al., J. Appl. Cryst. 26: 283-291 (1993)).

The structural and chemical similarities between DON and DONPEP.2peptide model were analyzed using POWERFIT (Microsimulations Inc.,Mahwah, N.J.), which tests a large number of random three-dimensionalalignments of two structures by using a Monte Carlo search algorithm.The quality of the superpositions was scored by using a complementaryfunction measuring van der Waals and electrostatic overlap between thetwo structures. The three-dimensional structure of nivalenol (CambridgeStructural Database (CSD) code: DUTJOR10), which was obtained from theCSD (Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge,United Kingdom, CB2 1EZ), was used in place of DON for the superpositionbecause a three-dimensional structure for DON is not available.Nivalenol binds to mAB 6F5 with slightly less affinity than DON binds tomAB 6F5. The side chain angles of the S, W, and F amino acid residues inthe peptide were allowed to rotate during POWERFIT superposition, andonly superpositions with the stereochemically acceptable side chainconformation were accepted. No bonds in the rigid nivalenol structurewere allowed to rotate. The results of the alignment were analyzedvisually with INSIGHT II.

Within the set of nonhomologous protein structures, 31 analogs ofDONPEP.2 tetrapeptides were found. The sequence search was limited tofour residues because close sequence matches to more than four residuesare rarely found in the Brookhaven PDB. The secondary structures ofthese tetrapeptides, which were assigned based on close backbonesuperposition with ideal secondary structures (α-helices, β-strands,reverse turns), were predominantly in the β-strand configuration (Table1).

To construct a three-dimensional structural model of SWGPFPF fromtetramer analogs in Table 1, we began by superimposing the analogs forresidues 1 to 4 (SWGP) were superimposed, which all formed a β-strand.The superpositions for residues 2 to 5 (WGPF) and 3 to 6 (GPFP)indicated that the central region of the peptide could for either aβ-strand or a reverse turn. However, the pair of proline residues in thesequence resulted in a significant kink even in the β-strand-likeanalogs (all of which had a superpositional RMSD equal to or greaterthan 0.9 Å in the GPFP region). Similarly, in the turn-forming analogs,the two proline residues made it impossible to form an ideal reverseturn. Thus, it seems likely that the central region forms a loose,inverted U-shaped turn flanked by residues in the β-strand conformation,which is prevalent in the analogs for residues 4 to 7 (shown in Table1).

TABLE 1 Secondary structure assignment of DONPEP.2 sequence analogs*.Confor- Backbone PDB Residue Analog mation RMSD (Å) Code Chain RangeSWGP DON mimotope peptide 1-4 SWGS Strand 0.580 Penicillinamidohydrolase 1 pnk B 64-67 SWGT Strand 1.264 Sulfhydryl proteinase 9pap 176-179 SWGT Strand 0.946 Xylanase 1 xnb 84-87 TWGP Strand 0.794Bluetongue virus coat protein 1 bvp 1 118-121 SFGT Strand 0.734Creatinase 1 chm A 325-328 SFGT Strand 0.453 Phosphotransferase 3 pmg A377-380 WGPF DON mimotope peptide 2-5 FGSF Turn 0.897 Fe(III) superoxidedismutase 1 isc A 100-103 FGSY Strand 0.465 Beta-lactamase 2 blt A322-325 FGSY Strand 0.730 Flavodoxin 4 fxn 85-88 FGTY Strand 0.530Simian virus 40 coat protein 1 sva 1 221-224 WGTY Turn 0.412 Xylanase 1xnb 85-88 FGPW Strand 0.956 Vitelline membrane protein 1 1 vmo A 126-129WGTW Turn 0.820 Glycosyl transferase 1 bpl B 215-218 WGTW Strand 0.660Vitelline membrane protein 1 1 vmo A 70-73 GPFP DON mimotope peptide 3-6GPFP Strand 1.080 Adenylosuccinate synthetase 1 ade A 276-279 GPFP Turn0.821 N-cadherin 1 nch A 15-18 GPFP Turn 0.518 Photosynthetic reactioncenter 1 pcr H 54-57 GPFT Turn 0.966 Aconitase 8 acn 324-327 GPFT Turn0.702 Monellin 1 mol A  9-12 GPYP Strand 0.909 Dioxygenase 2 pcd M445-448 GTFP Strand 1.110 Black beetle virus coat protein 2 bbv C131-134 GTFP Turn 0.732 Glycosyl transferase 1 xyz A 806-809 PFPF DONmimotope peptide 4-7 PFSF Strand 1.119 N-acethlneuraminate lyase 1 nal 1112-115 PFSY Turn 0.888 Acid phosphatase 1 kbp A 218-221 PFTY Strand1.127 Dialkyglycine decarboxylase 2 dkb 170-173 PYSY Turn 0.528Transthyretin 1 ttb A 113-116 PYTF Strand 0.599 Dethiobiotin synthase 1dts 73-76 PYTF Strand 0.498 Zinc endopeptidase 1 iae 16-19 PYTF Strand0.554 Acid phosphatase 1 kbp A 127-130 TYPY Turn 0.952 Adenylosuccinatesynthetase 1 ade A 234-237 TYPY Strand 1.086 Sulfhydryl proteinase 9 pap85-88 *Backbone superpositional RMSD values based on least squares fitof analogs of the DON mimetic peptide, SWGPFPF, onto regular secondarystructure templates. Each analog is assigned the conformation of thetemplate with which it has the lowest backbone superpositional RMSD. Theanalogs for SWGP start in strand conformation, then show a preferencefor forming a turn starting at the glycine residue. The last section ofthe peptide analogs favor strand conformations.

The conformation of the turn shown in FIG. 1C is based on superpositionof the turn forming analogs for residues 2 to 5.

The similarity between DON and the SWGPFPF model was determined by usingPOWERFIT to evaluate favorable three-dimensional superposition ofnivalenol (FIGS. 1B and 1D), a close analog of DON (FIG. 1A), onto thepeptide model. Ten independent superpositions, beginning with randomconformations, were performed by using POWERFIT. Three of the tenstructures with low superpositional energies showed a preference for theDON analog to align in a specific position along the peptide backbone ofthe model in the region spanning from the second-residue (tryptophan)main-chain nitrogen to the fifth-residue (phenylalanine) carbonyl carbon(FIG. 1E). The remaining superpositions showed a preference fornivalenol to align along the peptide backbone as well, although italigned with shorter sections. Also, side chain atoms of the secondresidue (tryptophan) in SWGPFPF overlapped with atoms of nivalenol inseveral of the superposition results.

EXAMPLE 10

A radio immunoassay (RIA) is performed using the C430 as follows. TheRIA procedure involves incubation of monoclonal antibody mAB 6F5simultaneously with a solution of unknown sample or known standard, anda constant amount of radioactively labled C430. After separation of thefree from bound C430, the radioactivity in the respective fractions isdetermined. The concentration of DON in the unknown sample is determinedby comparing results to a standard curve. Several known methods,including the ammonium sulfate precipitation method, double antibodytechnique, solid phase RIA method in which the immunoglobulin G (IgG) isconjugated to CNBr-activated SEPHAROSE gel (Pharmacia Biotech), adextran-coated charcoal colum and albumen-coated charcoal, is used forthe separation of free and bound C430 in RIA. A preferred method is anammonium precipitation method performed according to F. Chu et al.,Appl. Environ. Microbiol. 37: 104-108 (1979). In general, 50 μl ofradioactive C430 (10,000 to 15,000 dpm) is incubated with 0.15 ml ofanti-DON mAb solution of various dilutions in phosphate buffer (0.1 M,pH 7.2) at room temperature for 30 minutes, and then at 60° C.overnight. Separation of the bound from the free C430 is achieved by anammonium sulfate precipitation method according to Chu et al. (ibid.).Radioactivity is determined in a liquid scintillation counter in 5 ml ofAQUASOL (a product of New England Nuclear Corp., Boston, Mass.) foraqueous solutions.

EXAMPLE 11

This example provides an RIA assay for DON in a wheat sample usingradioactively labeled C430. DON is extracted from the wheat sample withacetonitrile:water (84:16), defatted with hexane, and reacted withacetic anhydride in pyridine to form DON-triacetate. The reactionmixture is loaded onto a C-18 cartridge to remove excess reagents andimpurities. Acetylated DON is eluted from the cartridge with 50%methanol solution, and analyzed by RIA using mAB 6F5 and radioactivelylabeled C430.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

8 1 21 DNA Artificial Sequence Description of Artificial SequenceSequence encoding the DONPEP.2 peptide mimotope of deoxynivalenol 1agttggggtc cttttccgtt t 21 2 7 PRT Artificial Sequence Description ofArtificial Sequence DONPEP.2 peptide mimotope of deoxynivalenol 2 SerTrp Gly Pro Phe Pro Phe 1 5 3 21 DNA Artificial Sequence Description ofArtificial Sequence Sequence encoding the DONPEP.12 peptide mimotope ofdeoxynivalenol 3 tcttggggtc cgcttccttt t 21 4 7 PRT Artificial SequenceDescription of Artificial Sequence DONPEP.12 peptide mimotope ofdeoxynivalenol 4 Ser Trp Gly Pro Leu Pro Phe 1 5 5 12 PRT ArtificialSequence Description of Artificial Sequence C430, the DONPEP.2 with astructurally flexible linker and a cysteine residue 5 Ser Trp Gly ProPhe Pro Phe Gly Gly Gly Ser Cys 1 5 10 6 7 PRT Artificial SequenceDescription of Artificial Sequence A consensus sequence for peptidemimotopes of deoxynivalenol 6 Ser Trp Gly Pro Xaa Pro Xaa 1 5 7 34 DNAArtificial Sequence Description of Artificial Sequence Sense primer 7gccaagctta gatcttggag cctttttttt ggag 34 8 34 DNA Artificial SequenceDescription of Artificial Sequence Antisense primer 8 ccggtcgacctgtatgggat tttgctaaac aact 34

We claim:
 1. In a method for determining whether a sample containsdeoxynivalenol (DON) in a competitive antibody binding assay whichcomprises providing a monoclonal antibody against the DON, reacting themonoclonal antibody with a limiting dilution series of the sample inreaction mixtures containing labeled DON as a competitor for a timesufficient for the DON or labeled DON as a competitor to bind themonoclonal antibody, separating the monoclonal antibody bound to the DONor labeled DON from the unbound DON or labeled DON in the reactionmixtures, and determining whether the sample contains the DON bydetecting the amount of labeled DON bound to the antibody in thereaction mixtures wherein detecting the labeled DON bound to themonoclonal antibody in the reaction mixtures indicates the sample doesnot contain DON, the improvement comprises providing as the competitor apeptide mimotope comprising amino acid sequence SWGPFPF as set forth inSeq. Id. No.2 which is bound by the monoclonal antibody.
 2. The methodof claim 1 wherein the peptide mimotope is conjugated to a reporter foran immunological assay wherein the reporter is selected from the groupconsisting of alkaline phosphatase, horseradish peroxidase, orfluorescence molecule.
 3. The method of claim 1 wherein the peptidemimotope is a part of a peptide or polypeptide.
 4. The method of claim 3wherein the polypeptide is selected from the group consisting ofalkaline phosphatase and horseradish peroxidase.
 5. The method of claim1 wherein the monoclonal antibody is mAB 6F5.
 6. A method fordetermining whether a sample contains deoxynivalenol (DON) whichcomprises: (a) incubating in a reaction mixture the sample, a monoclonalantibody against the DON, and a peptide mimotope comprising amino acidsequence SWGPFPF as set forth in Seq. Id. No.2 which is a competitor ofthe DON for the monoclonal antibody for a time sufficient for themonoclonal antibody to form a complex consisting of the DON or thepeptide mimotope of the DON; (b) separating the complex consisting ofthe DON bound by the monoclonal antibody and the complex formed by thepeptide mimotope and monoclonal antibody from the reaction; (c)detecting the complex consisting of the DON bound by the monoclonalantibody and the complex formed by the peptide mimotope and monoclonalantibody; and (d) comparing an amount of each of the complexes wherein adecrease in the amount of the complex comprising the peptide mimotopeindicates the sample contains the DON.
 7. The method of claim 6 whereinthe monoclonal antibody is produced by hybridoma cell line 6F5.
 8. Themethod of claim 6 wherein the peptide mimotope is conjugated to areporter for an immunological assay wherein the reporter is selectedfrom the group consisting of alkaline phosphatase, horseradishperoxidase, and fluorescence molecule.
 9. The method of claim 6 whereinthe peptide mimotope is a part of a peptide or polypeptide.
 10. Themethod of claim 9 wherein the polypeptide is selected from the groupconsisting of alkaline phosphatase and horseradish peroxidase.
 11. A kitfor determining whether a sample contains deoxynivalenol (DON)comprising: (a) a monoclonal antibody against the DON; (b) a peptidemimotope comprising amino acid sequence SWGPFPF as set forth in Seq. ID.No.2, which is a competitor of the DON for binding to the monoclonalantibody; and (c) instructions for using the kit.
 12. The kit of claim11 wherein the monoclonal antibody is produced by hybridoma cell line6F5.
 13. The kit of claim 11 wherein the peptide mimotope is conjugatedto a reporter for an immunological assay wherein the reporter isselected from the group consisting of alkaline phosphatase, horseradishperoxidase, and fluorescence molecule.
 14. The kit of claim 11 whereinthe peptide mimotope is a part of a peptide or polypeptide.
 15. The kitof claim 14 wherein the polypeptide is selected from the groupconsisting of alkaline phosphatase and horseradish peroxidase.